Taxonomical and functional composition of strawberry microbiome is genotype-dependent.

INTRODUCTION: Specific microbial communities are associated to host plants, influencing their phenotype and fitness.Despite the rising interest in plant microbiome, the role of microbial communities associated with perennial fruit plants remains overlooked. OBJECTIVES: This work provides the first comprehensive descriptionof the taxonomical and functional bacterial and fungal microbiota of below- and above-ground organsof three commercially important strawberry genotypes under cultural conditions. METHODS: Strawberry-associatedfungal and bacterial microbiomes were characterised by Next-Generation Sequencing and the potential functions expressed by the bacterial microbiome were analysed by both in silico and in vitro characterisation of plant growth-promoting abilities of native bacteria. Additionally, the association between the strawberry microbiome, plant disease tolerance, plant mineral nutrient content, and fruit quality was investigated. RESULTS: Results showed that thestrawberry core microbiome included 24 bacteria and 15 fungal operational taxonomicunits (OTUs).However, plant organ and genotype had a significant role in determining the taxonomical and functional composition of microbial communities. Interestingly, the cultivar with the highesttolerance against powdery mildew and leaf spot and the highest fruit productivity was the only one able to ubiquitously recruit the beneficial bacterium, Pseudomonasfluorescens, and to establish a mutualistic symbiosis with the arbuscular mycorrhizaRhizophagus irregularis. CONCLUSION: This work sheds light on the interaction of cultivated strawberry genotypes with a variety of microbes and highlights the importance of their applications to increase the sustainability of fruit crop production.

Red and Blue Light Differently Influence Actinidia chinensis Performance and Its Interaction with Pseudomonas syringae pv. Actinidiae.

Light composition modulates plant growth and defenses, thus influencing plant-pathogen interactions. We investigated the effects of different light-emitting diode (LED) red (R) (665 nm) and blue (B) (470 nm) light combinations on Actinidia chinensis performance by evaluating biometric parameters, chlorophyll a fluorescence, gas exchange and photosynthesis-related gene expression. Moreover, the influence of light on the infection by Pseudomonas syringae pv. actinidiae (Psa), the etiological agent of bacterial canker of kiwifruit, was investigated. Our study shows that 50%R-50%B (50R) and 25%R-75%B (25R) lead to the highest PSII efficiency and photosynthetic rate, but are the least effective in controlling the endophytic colonization of the host by Psa. Monochromatic red light severely reduced ΦPSII, ETR, Pn, TSS and photosynthesis-related genes expression, and both monochromatic lights lead to a reduction of DW and pigments content. Monochromatic blue light was the only treatment significantly reducing disease symptoms but did not reduce bacterial endophytic population. Our results suggest that monochromatic blue light reduces infection primarily by modulating Psa virulence more than host plant defenses.

Does Organic Farming Increase Raspberry Quality, Aroma and Beneficial Bacterial Biodiversity?

Plant-associated microbes can shape plant phenotype, performance, and productivity. Cultivation methods can influence the plant microbiome structure and differences observed in the nutritional quality of differently grown fruits might be due to variations in the microbiome taxonomic and functional composition. Here, the influence of organic and integrated pest management (IPM) cultivation on quality, aroma and microbiome of raspberry (Rubus idaeus L.) fruits was evaluated. Differences in the fruit microbiome of organic and IPM raspberry were examined by next-generation sequencing and bacterial isolates characterization to highlight the potential contribution of the resident-microflora to fruit characteristics and aroma. The cultivation method strongly influenced fruit nutraceutical traits, aroma and epiphytic bacterial biocoenosis. Organic cultivation resulted in smaller fruits with a higher anthocyanidins content and lower titratable acidity content in comparison to IPM berries. Management practices also influenced the amounts of acids, ketones, aldehydes and monoterpenes, emitted by fruits. Our results suggest that the effects on fruit quality could be related to differences in the population of Gluconobacter, Sphingomonas, Rosenbergiella, Brevibacillus and Methylobacterium on fruit. Finally, changes in fruit aroma can be partly explained by volatile organic compounds (VOCs) emitted by key bacterial genera characterizing organic and IPM raspberry fruits.

Bacterial volatile compound-based tools for crop management and quality.

Bacteria produce a huge diversity of metabolites, many of which mediate ecological relations. Among these, volatile compounds cause broad-range effects at low doses and, therefore, may be exploited for plant defence strategies and agricultural production, but such applications are still in their early development. Here, we review the latest technologies involving the use of bacterial volatile compounds for phytosanitary inspection, biological control, plant growth promotion, and crop quality. We highlight a variety of effects with a potential applicative interest, based on either live biocontrol and/or biostimulant agents, or the isolated metabolites responsible for the interaction with hosts or competitors. Future agricultural technologies may benefit from the development of new analytical tools to understand bacterial interactions with the environment.

A Breach in Plant Defences: Pseudomonas syringae pv. actinidiae Targets Ethylene Signalling to Overcome Actinidia chinensis Pathogen Responses.

Ethylene interacts with other plant hormones to modulate many aspects of plant metabolism, including defence and stomata regulation. Therefore, its manipulation may allow plant pathogens to overcome the host's immune responses. This work investigates the role of ethylene as a virulence factor for Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of the bacterial canker of kiwifruit. The pandemic, highly virulent biovar of this pathogen produces ethylene, whereas the biovars isolated in Japan and Korea do not. Ethylene production is modulated in planta by light/dark cycle. Exogenous ethylene application stimulates bacterial virulence, and restricts or increases host colonisation if performed before or after inoculation, respectively. The deletion of a gene, unrelated to known bacterial biosynthetic pathways and putatively encoding for an oxidoreductase, abolishes ethylene production and reduces the pathogen growth rate in planta. Ethylene production by Psa may be a recently and independently evolved virulence trait in the arms race against the host. Plant- and pathogen-derived ethylene may concur in the activation/suppression of immune responses, in the chemotaxis toward a suitable entry point, or in the endophytic colonisation.

Pseudomonas syringae pv. actinidiae: Ecology, Infection Dynamics and Disease Epidemiology.

Since 2008, the kiwifruit industry has been devastated by a pandemic outbreak of Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker. This disease has become the most significant limiting factor in kiwifruit production. Psa colonizes different organs of the host plant, causing a specific symptomatology on each of them. In addition, the systemic invasion of the plant may quickly lead to plant death. Despite the massive risk that this disease poses to the kiwifruit industry, studies focusing on Psa ecology have been sporadic, and a comprehensive description of the disease epidemiology is still missing. Optimal environmental conditions for infection, dispersal and survival in the environment, or the mechanisms of penetration and colonization of host tissues have not been fully elucidated yet. The present work aims to provide a synthesis of the current knowledge, and a deeper understanding of the epidemiology of kiwifruit bacterial canker based on new experimental data. The pathogen may survive in the environment or overwinter in dormant tissues and be dispersed by wind or rain. Psa was observed in association with several plant structures (stomata, trichomes, lenticels) and wounds, which could represent entry points for apoplast infection. Environmental conditions also affect the bacterial colonization, with lower optimum values of temperature and humidity for epiphytic than for endophytic growth, and disease incidence requiring a combination of mild temperature and leaf wetness. By providing information on Psa ecology, these data sets may contribute to plan efficient control strategies for kiwifruit bacterial canker.

N-Acyl Homoserine Lactones and Lux Solos Regulate Social Behaviour and Virulence of Pseudomonas syringae pv. actinidiae.

The phyllosphere is a complex environment where microbes communicate through signalling molecules in a system, generally known as quorum sensing (QS). One of the most common QS systems in Gram-negative proteobacteria is based on the production of N-acyl homoserine lactones (AHLs) by a LuxI synthase and their perception by a LuxR sensor. Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of the bacterial canker of kiwifruit, colonises plant phyllosphere before penetrating via wounds and natural openings. Since Psa genome encodes three LuxR solos without a cognate LuxI, this bacterium may perceive diffusible signals, but it cannot produce AHLs, displaying a non-canonical QS system. The elucidation of the mechanisms underlying the perception of environmental cues in the phyllosphere by this pathogen and their influence on the onset of pathogenesis are of crucial importance for a long-lasting and sustainable management of the bacterial canker of kiwifruit. Here, we report the ability of Psa to sense its own population density and the presence of surrounding bacteria. Moreover, we show that Psa can perceive AHLs, indicating that AHL-producing neighbouring bacteria may regulate Psa virulence in the host. Our results suggest that the ecological environment is important in determining Psa fitness and pathogenic potential. This opens new perspectives in the use of more advanced biochemical and microbiological tools for the control of bacterial canker of kiwifruit.

Biological control of bacterial plant diseases with Lactobacillus plantarum strains selected for their broad-spectrum activity.

The use of lactic acid bacteria (LAB) to control multiple pathogens that affect different crops was studied, namely, Pseudomonas syringae pv. actinidiae in kiwifruit, Xanthomonas arboricola pv. pruni in Prunus and Xanthomonas fragariae in strawberry. A screening procedure based on in vitro and in planta assays of the three bacterial pathogens was successful in selecting potential LAB strains as biological control agents. The antagonistic activity of 55 strains was first tested in vitro and the strains Lactobacillus plantarum CC100, PM411 and TC92, and Leuconostoc mesenteroides CM160 and CM209 were selected because of their broad-spectrum activity. The biocontrol efficacy of the selected strains was assessed using a multiple-pathosystem approach in greenhouse conditions. L. plantarum PM411 and TC92 prevented all three pathogens from infecting their corresponding plant hosts. In addition, the biocontrol performance of PM411 and TC92 was comparable to the reference products (Bacillus amyloliquefaciens D747, Bacillus subtilis QST713, chitosan, acibenzolar-S-methyl, copper and kasugamycin) in semi-field and field experiments. The in vitro inhibitory mechanism of PM411 and TC92 is based, at least in part, on a pH lowering effect and the production of lactic acid. Moreover, both strains showed similar survival rates on leaf surfaces. PM411 and TC92 can easily be distinguished because of their different multilocus sequence typing and random amplified polymorphic DNA profiles.

Pathogen-induced changes in floral scent may increase honeybee-mediated dispersal of Erwinia amylovora.

Honeybees are well recognised for their key role in plant reproduction as pollinators. On the other hand, their activity may vector some pathogens, such as the bacterium Erwinia amylovora, the causative agent of fire blight disease in pomaceous plants. In this research, we evaluated whether honeybees are able to discriminate between healthy and E. amylovora-infected flowers, thus altering the dispersal of the pathogen. For this reason, honeybees were previously trained to forage either on inoculated or healthy (control) apple flower. After the training, the two honeybee groups were equally exposed to inoculated and control flowering apple plants. To assess their preference, three independent methods were used: (1) direct count of visiting bees per time frame; (2) incidence on apple flowers of a marker bacterium (Pantoea agglomerans, strain P10c) carried by foragers; (3) quantification of E. amylovora populations in the collected pollen loads, proportional to the number of visits to infected flowers. The results show that both honeybee groups preferred control flowers over inoculated ones. The characterisation of volatile compounds released by flowers revealed a different emission of several bioactive compounds, providing an explanation for honeybee preference. As an unexpected ecological consequence, the influence of infection on floral scent increasing the visit rate on healthy flowers may promote a secondary bacterial spread.

Pathways of flower infection and pollen-mediated dispersion of Pseudomonas syringae pv. actinidiae, the causal agent of kiwifruit bacterial canker.

Flowers can provide a protected and nutrient-rich environment to the epiphytic microflora, thus representing a sensible entry point for pathogens such as Pseudomonas syringae pv. actinidiae (Psa). This bacterium can colonize both male and female Actinidia flowers, causing flower browning and fall, and systemic invasion of the host plant, eventually leading to its death. However, the process of flower colonization and penetration into the host tissues has not yet been fully elucidated. In addition, the presence of Psa in the pollen from infected flowers, and the role of pollination in the spread of Psa requires confirmation. The present study employed a Psa strain constitutively expressing the fluorescent GFPuv protein, to visualize in vivo flower colonization. Microscopy observations were performed by means of confocal laser scanning and wide-field fluorescent microscopy, and were coupled with the study of Psa population dynamics by quantitative PCR (q-PCR). The pathogen was shown to colonize stigmata, move along the stylar furrow, and penetrate the receptacles via the style or nectarhodes. Once the receptacle was invaded, the pathogen migrated along the flower pedicel and became systemic. Psa was also able to colonize the anthers epiphytically and endophytically. Infected male flowers produced contaminated pollen, which could transmit Psa to healthy plants. Finally, pollinators (Apis mellifera and Bombus terrestris) were studied in natural conditions, showing that, although they can be contaminated with Psa, the pathogen's transmission via pollinators is contrasted by its short survival in the hive.

Plant Microbiome and Its Link to Plant Health: Host Species, Organs and Pseudomonas syringae pv. actinidiae Infection Shaping Bacterial Phyllosphere Communities of Kiwifruit Plants.

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of the bacterial canker, the most devastating disease of kiwifruit vines. Before entering the host tissues, this pathogen has an epiphytic growth phase on kiwifruit flowers and leaves, thus the ecological interactions within epiphytic bacterial community may greatly influence the onset of the infection process. The bacterial community associated to the two most important cultivated kiwifruit species, Actinidia chinensis and Actinidia deliciosa, was described both on flowers and leaves using Illumina massive parallel sequencing of the V3 and V4 variable regions of the 16S rRNA gene. In addition, the effect of plant infection by Psa on the epiphytic bacterial community structure and biodiversity was investigated. Psa infection affected the phyllosphere microbiome structures in both species, however, its impact was more pronounced on A. deliciosa leaves, where a drastic drop in microbial biodiversity was observed. Furthermore, we also showed that Psa was always present in syndemic association with Pseudomonas syringae pv. syringae and Pseudomonas viridiflava, two other kiwifruit pathogens, suggesting the establishment of a pathogenic consortium leading to a higher pathogenesis capacity. Finally, the analyses of the dynamics of bacterial populations provided useful information for the screening and selection of potential biocontrol agents against Psa.

Insecticidal Activity of Photorhabdus luminescens against Drosophila suzukii.

Drosophila suzukii causes considerable economic damage to small and thin-skinned fruits including cherry, blueberry, raspberry, grape and strawberry. Since it attacks fruits at the ripening stage, the use of chemical pesticides is limited due to the high risk of residues on fruit. Biological control is thus expected to play an essential role in managing this pest. The Gram-negative bacterium, Photorhabdus luminescens and its symbiotic Heterorhabditis spp. nematode have been shown to be highly pathogenic to insects, with a potential for replacing pesticides to suppress several pests. Insecticidal activity of P. luminescens at different bacterial cell concentrations and its cell-free supernatant were assessed against third-instar larvae and pupae of D. suzukii under laboratory conditions. P. luminescens suspensions had a significant oral and contact toxicity on D. suzukii larvae and pupae, with mortalities up to of 70⁻100% 10 days after treatment. Cell-free supernatant in the diet also doubled mortality rates of feeding larvae. Our results suggest that P. luminescens may be a promising candidate for biological control of D. suzukii, and its use in integrated pest management (IPM) programs is discussed.

Comparative transcriptome analysis of the interaction between Actinidia chinensis var. chinensis and Pseudomonas syringae pv. actinidiae in absence and presence of acibenzolar-S-methyl.

BACKGROUND: Since 2007, bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has become a pandemic disease leading to important economic losses in every country where kiwifruit is widely cultivated. Options for controlling this disease are very limited and rely primarily on the use of bactericidal compounds, such as copper, and resistance inducers. Among the latter, the most widely studied is acibenzolar-S-methyl. To elucidate the early molecular reaction of kiwifruit plants (Actinidia chinensis var. chinensis) to Psa infection and acibenzolar-S-methyl treatment, a RNA seq analysis was performed at different phases of the infection process, from the epiphytic phase to the endophytic invasion on acibenzolar-S-methyl treated and on non-treated plants. The infection process was monitored in vivo by confocal laser scanning microscopy. RESULTS: De novo assembly of kiwifruit transcriptome revealed a total of 39,607 transcripts, of which 3360 were differentially expressed during the infection process, primarily 3 h post inoculation. The study revealed the coordinated changes of important gene functional categories such as signaling, hormonal balance and transcriptional regulation. Among the transcription factor families, AP2/ERF, MYB, Myc, bHLH, GATA, NAC, WRKY and GRAS were found differentially expressed in response to Psa infection and acibenzolar-S-methyl treatment. Finally, in plants treated with acibenzolar-S-methyl, a number of gene functions related to plant resistance, such as PR proteins, were modulated, suggesting the set-up of a more effective defense response against the pathogen. Weighted-gene coexpression network analysis confirmed these results. CONCLUSIONS: Our work provides an in-depth description of the plant molecular reactions to Psa, it highlights the metabolic pathway related to acibenzolar-S-methyl-induced resistance and it contributes to the development of effective control strategies in open field.

Role of Metcalfa pruinosa as a Vector for Pseudomonas syringae pv. actinidiae.

After 20 years of steady increase, kiwifruit industry faced a severe arrest due to the pandemic spread of the bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa). The bacterium penetrates the host plant primarily via natural openings or wounds, and its spread is mainly mediated by atmospheric events and cultural activities. Since the role of sucking insects as vectors of bacterial pathogens is widely documented, we investigated the ability of Metcalfa pruinosa Say (1830), one of the most common kiwifruit pests, to transmit Psa to healthy plants in laboratory conditions. Psa could be isolated both from insects feeding over experimentally inoculated plants, and from insects captured in Psa-infected orchards. Furthermore, insects were able to transmit Psa from experimentally inoculated plants to healthy ones. In conclusion, the control of M. pruinosa is recommended in the framework of protection strategies against Psa.

Evaluation of Penicillium expansum isolates for aggressiveness, growth and patulin accumulation in usual and less common fruit hosts.

Experiments were carried out in vivo and in vitro with four isolates of Penicillium expansum (I 1, E 11, C 28 and I 12) to evaluate their aggressiveness, growth and patulin accumulation in both usual (pears and apples) and less common hosts (apricots, peaches, strawberries and kiwifruits) of the pathogen. The 75% of isolates showed the ability to cause blue mould in all tested hosts. In particular, C 28 and I 1 were the most and the least aggressive isolates, respectively (52.9 and 10.6% infection and 20.7 and 15.4 mm lesion diameters). 'Candonga' strawberries and 'Pinkcot' apricots showed the largest lesion diameters (29.8 and 25.3 mm), followed by 'Conference' pears, 'Spring Crest' peaches and 'Abate Fetel' pears. With the exception of 'Candonga' strawberries, the formation of colonies and mycelial growth of P. expansum isolates on fruit puree agar media (PAMs) was stimulated in comparison to a standard growth medium (malt extract agar, MEA). Two of the most aggressive isolates in our assays (I 12 and C 28) showed the greatest accumulation of patulin both in vitro and in vivo, while the least aggressive isolate (I 1) produced patulin only in a few growth media and cvs. Patulin concentration on fruit PAMs was higher than patulin detected in infected fruit tissues. Apple PAMs were the more favorable substrates for patulin accumulation in vitro (maximum concentration 173.1 and 74.1 µg/mL in 'Pink Lady and 'Golden Delicious' PAMs, respectively) and 'Pink Lady' apples inoculated with the isolate E 11 showed the greatest accumulation of patulin in the whole in vivo assay (33.9 µg/mL). However, infected tissue of cv Golden Delicious showed lower average accumulation of patulin (1.7 µg/mL) than that of cv Pink Lady (19.1 µg/mL), and no significant differences in patulin concentrations were found among 'Golden Delicious' apples and tested cvs of pears, kiwifruits and strawberries. Peaches were highly susceptible to patulin accumulation, showing average concentrations of 27.4 and 18.6 µg/mL in vitro and in vivo, respectively. Apricots were also consistently positive for patulin accumulation, both in vitro (average values of 20.1 µg/mL) and in vivo (average values of 9.4 µg/mL). Our study showed the potential of some less common hosts of P. expansum (in particular peaches and apricots) to support patulin production, indicating that a steady monitoring of patulin contamination should be carried out in fruit substrates other than apples and pears.